The Region Preceding the C-Terminal NWETF Pentapeptide Modulates Baseline Activity and Aspartate Inhibition of Escherichia coli Tar

Abstract

The Tar chemoreceptor-CheA-CheW ternary complex of Escherichia coli is a transmembrane allosteric enzyme in which binding of ligands to the periplasmic domain modulates the activity of CheA kinase. Kinase activity is also affected by reversible methylation of four glutamyl residues in the cytoplasmic domain of the receptor. E. coli Tar contains 553 residues. Residues 549-553 comprise the NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. The crystal structure of the similar Tsr chemoreceptor predicts that residues 263-289 and 490-515 of Tar form the most membrane-proximal portion of the extended CD1-CD2 four-helix bundle of the cytoplasmic domain. The last methylation site, Glu-491, is in the C19 heptad, and the N22-19 and C22-19 heptads are present in all classes of bacterial transmembrane chemoreceptors. Residues 516-548 probably serve as a flexible tether for the NWETF pentapeptide. Here, we present a mutational analysis of residues 505-548. The more of this region that is deleted, the less sensitive Tar is to inhibition by aspartate. Tar deleted from residue 505 through the NWETF sequence stimulates CheA in vitro but is not inhibited by aspartate. Thus, interaction of the last two heptads (C21 and C22) of CD2 with the first two heptads (N22 and N21) of CD1 must be important for transmitting an inhibitory signal from the HAMP domain to the four-helix bundle. The R514A, K523A, R529A, R540A, and R542A substitutions, singly or together, increase the level of activation of CheA in vitro, whereas the R505A substitution decreases the level of CheA stimulation by 40% and lowers the aspartate K(i) 7-fold. The R505E substitution completely abolishes stimulation of CheA in vitro. Glu-505 may interact electrostatically with Asp-273 to destabilize the "on" signaling state by loosening the four-helix bundle.

A zipped-helix cap potentiates HAMP domain control of chemoreceptor signaling

“…Several in vitro studies, including hydrogen-exchange MS (57), NMR dynamics approaches (58), and electron paramagnetic resonance (59)(60)(61), have demonstrated differential dynamic properties of the MH1 and MH2 helices that are consistent with the zipped-cap model. The in vivo signaling properties of truncated Tar receptors are also consistent with the zipped-cap model (62).…”

Ligand Affinity and Kinase Activity Are Independent of Bacterial Chemotaxis Receptor Concentration: Insight into Signaling Mechanisms

“…Additional activity measurements on the 100:1 samples demonstrated that even 400 mM Ser did not inhibit kinase activation by Tsr 4Q proteoliposomes (40). The lack of Ser inhibition of Tsr 4Q might be related to the C-terminal deletion in our construct, since deletions of residues in the 502–515 range of the Asp receptor have been shown to interfere with Asp inhibition of kinase activity (43). However, our Tsr construct is intact through residue 517, and is capable of wild type levels of kinase inhibition, at least for the Tsr 2Q2E (31) and Tsr 4E (this study) methylation states.…”

Structural insight into the low affinity between Thermotoga maritima CheA and CheB compared to their Escherichia coli/Salmonella typhimurium counterparts

“…However, the apparent low binding affinity (K D > 1 mM) for T. maritima CheB and CheA doesn’t necessarily mean that these two proteins have a low probability of interaction in the context of the signaling complexes inside the bacterial cell. For instance, some chemoreceptors of E. coli and S. typhimurium contain a conserved pentapeptide sequence (NWETF) that binds to CheR and CheB [33, 34]. Although NWETF sequence is not conserved in T. maritima chemoreceptors [35], there is a possibility that T. maritima CheB is tethered to chemoreceptors in close proximity to CheA, thereby increasing the effective local concentrations of the two proteins.…”